During the past year, this laboratory continued studies on the mechanism of HIV entry and tropism, as well as development of therapeutic and protective strategies based on the molecules involved in entry. 1) Expansion of the coreceptor repertoire. Initially, we identified CXCR4 and CCR5 as major coreceptors. We have since shown that amongst diverse primary HIV-1 isolates, usage of these coreceptors correlates closely with tropism for T-cell lines vs. primary macrophages. We also found that many primary strains use CCR3, as well as the orphan receptor STRL33. Coreceptor activity has also been observed for CCR8; since CCR8 is abundantly expressed in thymus but not in peripheral T cells or macrophages, we are exploring the possibility that HIV might adapt to use this coreceptor when replicating in the thymus. We also demonstrated that another orphan is a receptor for fractalkine, the first member of the CX3C family of chemokines; CX3CR1 was found to have coreceptor activity for diverse HIV-1 strains. In the HL60 model for eosinophil differention, we demonstrated different modes of regulation for CCR3 and CCR1. 2) Molecular mechanism of fusion. We have expanded on our earlier demonstration that soluble CD4 activates Env to fuse with cells expressing coreceptor but not CD4. We have shown that soluble CD4 exposes new neutralization epitopes involved in coreceptor interaction. These findings have implications for new HIV vaccine strategies. We have also initiated efforts to define minimal regions of Env essential for interaction with coreceptors, as well as to define critical regions of the coreceptors, with particular emphasis on the importance of transmembrane regions. 3) Therapeutic strategies based on molecules involved in fusion. We analyzed a battery of compounds identified by the Division of AIDS as HIV inhibitors that act at an early step in the replication cycle. Several of these were found to be potent inhibitors of Env-mediated fusion. Each acted with comparable efficiency with different coreceptors; the molecular site of action of these inhibitors is an important question for their potential use as anti-HIV drugs or topical transmission-blocking agents. We also demonstrated fusion inhibition by synthetic peptides representing certain transmembrane regions of CXCR4 and CCR5. Finally we characterized a single-chain immunotoxin made with a monoclonal antibody having broad cross-reactivity against diverse HIV-1 strains; the immunotoxin showed selective cytotoxicity against HIV Env-expressing cells, with potency greater than that of CD4-Pseudomonas exotoxin. We are pursuing the possible utility of Env-targeted toxins for eliminating reservoirs of HIV-infected cells from people undergoing highly active antiretroviral therapy.